Cell Wall Integrity Signaling Pathway Research Articles

Transcriptional reporters for genes activated by cell wall stress through a non‐catalytic mechanism involving Mpk1 and SBF

The Mpk1 MAP kinase of the cell wall integrity (CWI) signalling pathway induces transcription of the FKS2 gene in response to cell wall stress through a non-catalytic mechanism that involves stable association of Mpk1 with the Swi4 transcription factor. This dimeric complex binds to a Swi4 recognition site in the FKS2 promoter. The Swi6 transcription factor is also required to bind this ternary complex for transcription initiation to ensue. In this context, the Mlp1 pseudokinase serves a redundant function with Mpk1. We have identified three additional genes, CHA1, YLR042c and YKR013w, that are induced by cell wall stress through the same mechanism. We report on the behaviour of several promoter-lacZ reporter plasmids designed to detect cell wall stress transcription through this pathway.

Yeast Mpk1 Cell Wall Integrity Mitogen-activated Protein Kinase Regulates Nucleocytoplasmic Shuttling of the Swi6 Transcriptional Regulator

The yeast SBF transcription factor is a heterodimer comprised of Swi4 and Swi6 that has a well defined role in cell cycle-specific transcription. SBF serves a second function in the transcriptional response to cell wall stress in which activated Mpk1 mitogen-activated protein kinase of the cell wall integrity signaling pathway forms a complex with Swi4, the DNA binding subunit of SBF, conferring upon Swi4 the ability to bind DNA and activate transcription of FKS2. Although Mpk1-Swi4 complex formation and transcriptional activation of FKS2 does not require Mpk1 catalytic activity, Swi6 is phosphorylated by Mpk1 and must be present in the Mpk1-Swi4 complex for transcriptional activation of FKS2. Here, we find that Mpk1 regulates Swi6 nucleocytoplasmic shuttling in a biphasic manner. First, formation of the Mpk1-Swi4 complex recruits Swi6 to the nucleus for transcriptional activation. Second, Mpk1 negatively regulates Swi6 by phosphorylation on Ser238, which inhibits nuclear entry. Ser238 neighbors a nuclear localization signal (NLS) whose function is blocked by phosphorylation at Ser238 in a manner similar to the regulation by Cdc28 of another Swi6 NLS, revealing a mechanism for the integration of multiple signals to a single endpoint. Finally, the Kap120 beta-importin binds the Mpk1-regulated Swi6 NLS but not the Cdc28-regulated NLS.

Mechanism of Mpk1 Mitogen-Activated Protein Kinase Binding to the Swi4 Transcription Factor and Its Regulation by a Novel Caffeine-Induced Phosphorylation

The Mpk1 mitogen-activated protein kinase (MAPK) of the cell wall integrity signaling pathway uses a noncatalytic mechanism to activate the SBF (Swi4/Swi6) transcription factor. Active Mpk1 forms a complex with Swi4, the DNA-binding subunit of SBF, conferring the ability to bind DNA. Because SBF activation is independent of Mpk1 catalytic activity but requires Mpk1 to be in an active conformation, we sought to understand how Mpk1 interacts with Swi4. Mutational analysis revealed that binding and activation of Swi4 by Mpk1 requires an intact D-motif-binding site, a docking surface common to MAPKs that resides distal to the phosphorylation loop but does not require the substrate-binding site, revealing a novel mechanism for MAPK target regulation. Additionally, we found that Mpk1 binds near the autoinhibitory C terminus of Swi4, suggesting an activation mechanism in which Mpk1 substitutes for Swi6 in promoting Swi4 DNA binding. Finally, we show that caffeine is an atypical activator of cell wall integrity signaling, because it induces phosphorylation of the Mpk1 C-terminal extension at Ser423 and Ser428. These phosphorylations were dependent on the DNA damage checkpoint kinases, Mec1/Tel1 and Rad53. Phosphorylation of Ser423 specifically blocked SBF activation by preventing Mpk1 association with Swi4, revealing a novel mechanism for regulating MAPK target specificity.

The MpkA MAP kinase module regulates cell wall integrity signaling and pyomelanin formation in Aspergillus fumigatus

Aspergillus fumigatus is the most important air-borne fungal pathogen, causing severe infections in immunocompromised patients. Mitogen-activated protein kinase (MAPK) signaling pathways are involved in the regulation of various cellular responses to environmental changes in eukaryotes. Genome Blast analysis revealed that the central core of the cell wall integrity signaling pathway in A. fumigatus is composed of three protein kinases designated Bck1, Mkk2 and MpkA. This pathway is of particular interest because it represents a possible target for new antifungal drugs. Deletion of these genes resulted in severe sensitivity of the mutants against cell wall-disturbing compounds and drastic alterations of the fungal morphology. Western blot analysis demonstrated that Bck1 and Mkk2 directly activate MpkA during vegetative growth and under cell wall stress conditions further confirming that Bck1, Mkk2 and MpkA form a MAP kinase module. Interestingly, this MAP kinase module affects the formation of pyomelanin derived from tyrosine degradation.

Deregulation of DSE1 Gene Expression Results in Aberrant Budding within the Birth Scar and Cell Wall Integrity Pathway Activation in Saccharomyces cerevisiae

Strains of Saccharomyces cerevisiae lacking Isw2, the catalytic subunit of the Isw2 chromatin remodeling complex, show the mating type-independent activation of the cell wall integrity (CWI) signaling pathway. Since the CWI pathway activation usually reflects cell wall defects, we searched for the cell wall-related genes changed in expression. The genes DSE1, CTS1, and CHS1 were upregulated as a result of the absence of Isw2, according to previously published gene expression profiles (I. Frydlova, M. Basler, P. Vasicova, I. Malcova, and J. Hasek, Curr. Genet. 52:87-95, 2007). Western blot analyses of double deletion mutants, however, did not indicate the contribution of the chitin metabolism-related genes CTS1 and CHS1 to the CWI pathway activation. Nevertheless, the deletion of the DSE1 gene encoding a daughter cell-specific protein with unknown function suppressed CWI pathway activation in isw2Delta cells. In addition, the deletion of DSE1 also abolished the budding-within-the-birth-scar phenotype of isw2Delta cells. The plasmid-driven overexpression proved that the deregulation of Dse1 synthesis was also responsible for CWI pathway activation and manifestation of the budding-within-the-birth-scar phenotype in wild-type cells. The overproduced Dse1-green fluorescent protein localized to both sides of the septum and persisted in unbudded cells. Although the exact cellular role of this daughter cell-specific protein has to be elucidated, our data point to the involvement of Dse1 in bud site selection in haploid cells.

The Synthetic Genetic Network around PKC1 Identifies Novel Modulators and Components of Protein Kinase C Signaling in Saccharomyces cerevisiae

Budding yeast Saccharomyces cerevisiae contains one protein kinase C (PKC) isozyme encoded by the essential gene PKC1. Pkc1 is activated by the small GTPase Rho1 and plays a central role in the cell wall integrity (CWI) signaling pathway. This pathway acts primarily to remodel the cell surface throughout the normal life cycle and upon various environmental stresses. The pathway is heavily branched, with multiple nonessential branches feeding into and out of the central essential Rho1-Pkc1 module. In an attempt to identify novel components and modifiers of CWI signaling, we determined the synthetic lethal genetic network around PKC1 by using dominant-negative synthetic genetic array analysis. The resulting mutants are hypersensitive to lowered Pkc1 activity. The corresponding 21 nonessential genes are closely related to CWI function: 14 behave in a chemical-genetic epistasis test as acting in the pathway, and 6 of these genes encode known components. Twelve of the 21 null mutants display elevated CWI reporter activity, consistent with the idea that the pathway is activated by and compensates for loss of the gene products. Four of the 21 mutants display low CWI reporter activity, consistent with the idea that the pathway is compromised in these mutants. One of the latter group of mutants lacks Ack1(Ydl203c), an uncharacterized SEL-1 domain-containing protein that we find modulates pathway activity. Epistasis analysis places Ack1 upstream of Pkc1 in the CWI pathway and dependent on the upstream Rho1 GTP exchange factors Rom2 and Tus1. Overall, the synthetic genetic network around PKC1 directly and efficiently identifies known and novel components of PKC signaling in yeast.

Yeast genomic expression patterns in response to low-shear modeled microgravity

The low-shear microgravity environment, modeled by rotating suspension culture bioreactors called high aspect ratio vessels (HARVs), allows investigation in ground-based studies of the effects of microgravity on eukaryotic cells and provides insights into the impact of space flight on cellular physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) causes significant phenotypic changes of a select group of Saccharomyces cerevisiae genes associated with the establishment of cell polarity, bipolar budding, and cell separation. However, the mechanisms cells utilize to sense and respond to microgravity and the fundamental gene expression changes that occur are largely unknown. In this study, we examined the global transcriptional response of yeast cells grown under LSMMG conditions using DNA microarray analysis in order to determine if exposure to LSMMG results in changes in gene expression.LSMMG differentially changed the expression of a significant number of genes (1372) when yeast cells were cultured for either five generations or twenty-five generations in HARVs, as compared to cells grown under identical conditions in normal gravity. We identified genes in cell wall integrity signaling pathways containing MAP kinase cascades that may provide clues to novel physiological responses of eukaryotic cells to the external stress of a low-shear modeled microgravity environment. A comparison of the microgravity response to other environmental stress response (ESR) genes showed that 26% of the genes that respond significantly to LSMMG are involved in a general environmental stress response, while 74% of the genes may represent a unique transcriptional response to microgravity. In addition, we found changes in genes involved in budding, cell polarity establishment, and cell separation that validate our hypothesis that phenotypic changes observed in cells grown in microgravity are reflected in genome-wide changes.This study documents a considerable response to yeast cell growth in low-shear modeled microgravity that is evident, at least in part, by changes in gene expression. Notably, we identified genes that are involved in cell signaling pathways that allow cells to detect environmental changes, to respond within the cell, and to change accordingly, as well as genes of unknown function that may have a unique transcriptional response to microgravity. We also uncovered significant changes in the expression of many genes involved in cell polarization and bud formation that correlate well with the phenotypic effects observed in yeast cells when grown under similar conditions. These results are noteworthy as they have implications for human space flight.

Characterization of GPI14/YJR013w mutation that induces the cell wall integrity signalling pathway and results in increased protein production in Saccharomyces cerevisiae

We report here identification and characterization of a mutation in the GPI14 gene, the yeast homologue of the mammalian PIG-M that functions in the synthesis of the GPI moiety anchoring proteins to the plasma membrane. We show that the first putative transmembrane domain of Gpi14p is not essential for its function. Downregulation of GPI14 expression/reduced protein function due to an amino terminal deletion resulted in increased transcription and production of an endogenous and a heterologous secreted protein expressed from HSP150 and ADH1 promoter, respectively. In these cells, unfolded protein response was induced but was not responsible for the enhanced production of these proteins. A cell wall defect in the gpi14 mutant cells was suggested by cell aggregation phenotype, increased sensitivity to Calcofluor white, an increased release of Gas1p and total protein into the culture medium. In the gpi14 mutant cells, transcription of RLM1, a transcription factor participating in the cell wall integrity signalling pathway, was increased, and deletion of RLM1 resulted in a synthetic lethal phenotype with the gpi14 mutation. These results suggest that partial inactivation of Gpi14p causes defects in the cell wall structure and suggest that compromised GPI anchor synthesis results in enhanced protein production via the cell wall integrity signalling pathway.

Cell Wall Integrity Signaling inSaccharomyces cerevisiae

The yeast cell wall is a highly dynamic structure that is responsible for protecting the cell from rapid changes in external osmotic potential. The wall is also critical for cell expansion during growth and morphogenesis. This review discusses recent advances in understanding the various signal transduction pathways that allow cells to monitor the state of the cell wall and respond to environmental challenges to this structure. The cell wall integrity signaling pathway controlled by the small G-protein Rho1 is principally responsible for orchestrating changes to the cell wall periodically through the cell cycle and in response to various forms of cell wall stress. This signaling pathway acts through direct control of wall biosynthetic enzymes, transcriptional regulation of cell wall-related genes, and polarization of the actin cytoskeleton. However, additional signaling pathways interface both with the cell wall integrity signaling pathway and with the actin cytoskeleton to coordinate polarized secretion with cell wall expansion. These include Ca(2+) signaling, phosphatidylinositide signaling at the plasma membrane, sphingoid base signaling through the Pkh1 and -2 protein kinases, Tor kinase signaling, and pathways controlled by the Rho3, Rho4, and Cdc42 G-proteins.

Rsp5 ubiquitin ligase affects isoprenoid pathway and cell wall organization in S. cerevisiae.

Dimethylallyl diphosphate, an isomer of isopentenyl diphosphate, is a common substrate of Mod5p, a tRNA modifying enzyme, and the farnesyl diphosphate synthase Erg20p, the key enzyme of the isoprenoid pathway. rsp5 mutants, defective in the Rsp5 ubiquitin-protein ligase, were isolated and characterized as altering the mitochondrial/cytosolic distribution of Mod5p. To understand better how competition for the substrate determines the regulation at the molecular level, we analyzed the effect of the rsp5-13 mutation on Erg20p expression. The level of Erg20p was three times lower in rsp5-13 compared to the wild type strain and this effect was dependent on active Mod5p. Northern blot analysis indicated a regulatory role of Rsp5p in ERG20 transcription. ERG20 expression was also impaired in pkc1Delta lacking a component of the cell wall integrity signaling pathway. Low expression of Erg20p in rsp5 cells was accompanied by low level of ergosterol, the main end product of the isoprenoid pathway. Additionally, rsp5 strains were resistant to nystatin, which binds to ergosterol present in the plasma membrane, and sensitive to calcofluor white, a drug destabilizing cell wall integrity by binding to chitin. Furthermore, the cell wall structure appeared abnormal in most rsp5-13 cells investigated by electron microscopy and chitin level in the cell wall was increased two-fold. These results indicate that Rsp5p affects the isoprenoid pathway which has important roles in ergosterol biosynthesis, protein glycosylation and transport and in this way may influence the composition of the plasma membrane and cell wall.

Rsp5 ubiquitin ligase affects isoprenoid pathway and cell wall organization in S. cerevisiae.

Dimethylallyl diphosphate, an isomer of isopentenyl diphosphate, is a common substrate of Mod5p, a tRNA modifying enzyme, and the farnesyl diphosphate synthase Erg20p, the key enzyme of the isoprenoid pathway. rsp5 mutants, defective in the Rsp5 ubiquitin-protein ligase, were isolated and characterized as altering the mitochondrial/cytosolic distribution of Mod5p. To understand better how competition for the substrate determines the regulation at the molecular level, we analyzed the effect of the rsp5-13 mutation on Erg20p expression. The level of Erg20p was three times lower in rsp5-13 compared to the wild type strain and this effect was dependent on active Mod5p. Northern blot analysis indicated a regulatory role of Rsp5p in ERG20 transcription. ERG20 expression was also impaired in pkc1Delta lacking a component of the cell wall integrity signaling pathway. Low expression of Erg20p in rsp5 cells was accompanied by low level of ergosterol, the main end product of the isoprenoid pathway. Additionally, rsp5 strains were resistant to nystatin, which binds to ergosterol present in the plasma membrane, and sensitive to calcofluor white, a drug destabilizing cell wall integrity by binding to chitin. Furthermore, the cell wall structure appeared abnormal in most rsp5-13 cells investigated by electron microscopy and chitin level in the cell wall was increased two-fold. These results indicate that Rsp5p affects the isoprenoid pathway which has important roles in ergosterol biosynthesis, protein glycosylation and transport and in this way may influence the composition of the plasma membrane and cell wall.

Mutational analysis of the cytoplasmic domain of the Wsc1 cell wall stress sensor.

Wsc1 is a member of a family of highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae and function as sensors of cell wall stress. These proteins activate the cell wall integrity signalling pathway by stimulating the small G-protein Rho1, protein kinase C (Pkc1) and a MAP kinase cascade. The cytoplasmic domains of Wsc1 family members interact with the Rom2 guanine nucleotide exchange factor to stimulate GTP-binding of Rho1. Here, a mutational analysis of the cytoplasmic domain of Wsc1 is presented. The data identify two regions of the Wsc1 cytoplasmic tail that are conserved with other family members as important for Rom2 interaction. These regions are separated by an inhibitory region, which includes a cluster of seryl residues that appear to be phosphorylated. Mutational analysis of these residues supports a model in which Wsc1 interaction with Rom2 is negatively regulated by phosphorylation.

Feedback Inhibition on Cell Wall Integrity Signaling by Zds1 Involves Gsk3 Phosphorylation of a cAMP-dependent Protein Kinase Regulatory Subunit

We report here that budding yeast cAMP-dependent protein kinase (cAPK) is controlled by heat stress. A rise in temperature from 30 to 37 degrees C was found to result in both a higher expression and an increased cytoplasmic localization of its regulatory subunit Bcy1. Both of these effects required phosphorylation of serines located in its localization domain. Surprisingly, classic cAPK-controlled processes were found to be independent of Bcy1 phosphorylation, indicating that these modifications do not affect cAPK activity as such. Alternatively, phosphorylation may recruit cAPK to, and thereby control, a specific subset of (perhaps novel) cAPK targets that are presumably localized extranuclearly. Zds1 and Zds2 may play a role in this process, since these were found required to retain hyperphosphorylated Bcy1 in the cytoplasm at 37 degrees C. Mck1, a homologue of mammalian glycogen synthase kinase 3 and a downstream component of the heat-activated Pkc1-Slt2/Mpk1 cell wall integrity pathway, is partly responsible for hyperphosphorylations of Bcy1. Remarkably, Zds1 appears to act as a negative regulator of cell wall integrity signaling, and this activity is dependent in part on the phosphorylation status of Bcy1. Thus, Mck1 phosphorylation of Bcy1 and Zds1 may constitute an unprecedented negative feedback control on the cell wall integrity-signaling pathway.

Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase.

The Mpk1 MAP kinase of the Saccharomyces cerevisiae cell wall integrity signalling pathway phosphorylates and activates the Rlm1 transcription factor in response to cell wall stress. Rlm1 is related to mammalian MEF2 isoforms, and shares a similar DNA-binding specificity. Signalling through Rlm1 regulates the expression of at least 25 genes, most of which have been implicated in cell wall biogenesis. We report here the transcriptional induction by agents of cell wall stress of a set of lacZ reporter plasmids derived from several Rlm1-responsive genes. Analysis of substitution mutations at putative Mpk1 phosphorylation sites within Rlm1 revealed that Ser427 and Thr439 are important for its stress-induced transcriptional activation of these reporter plasmids. Assessment of Rlm1 activation potency when fused to a heterologous DNA-binding domain showed that the identified seryl and threonyl residues are necessary for the Rlm1 transcriptional activation function independently of its DNA binding. We also demonstrate that a MAP kinase docking site, shown recently to mediate activation of MEF2A and MEF2C, is conserved in Rlm1 and is required for its ability to mediate transcriptional activation in response to agents that induce cell wall stress. Finally, intracellular localization analyses show that Rlm1 resides in the nucleus regardless of its activation and phosphorylation status. Together these observations support the inference that Mpk1 regulates the Rlm1 transcriptional activation function by phosphorylation of Ser427 and Thr439.

Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1.

Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2.

Genome-wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway.

The cell integrity pathway of Saccharomyces cerevisiae monitors cell wall remodelling during growth and differentiation. Additionally, this pathway responds to environmental stresses that challenge the integrity of the cell wall. We conducted a genome-wide survey of genes whose expression was altered in response to activation of Mpk1/Slt2, the MAP kinase, under the control of cell integrity signalling. We identified 25 genes whose regulation was altered by Mpk1 activity. Among these, 20 were positively regulated by Mpk1, and five were negatively regulated. Most of the genes identified encode either known or suspected cell wall proteins or enzymes involved in cell wall biogenesis. These include glycosyl-phosphatidylinositol (GPI) proteins, members of the Pir family of cell wall proteins, Mpk1 itself and others. All of the regulation detected was mediated by the Rlm1 transcription factor, a MADS-box protein that is phosphorylated and activated by Mpk1, but for which no transcriptional targets had been identified. A similar pattern of regulation was observed when cell integrity signalling was induced by environmental stress (i.e. temperature upshift).